WO2022081716A1 - Methods of treating iga nephropathy with thiol-containing molecules - Google Patents

Abstract

A method for treatment or preventing IgA nephropathy in a subject is provided. The method of treating or preventing IgA nephropathy includes administering a thiol-containing molecule, or a pharmaceutically acceptable salt thereof to a mammal in need thereof. Also provided is a method and kit for screening candidate therapeutic agents for treating or preventing IgA nephropathy.

Description

METHODS OF TREATING IgA NEPHROPATHY WITH THIOL-CONTAINING

MOLECULES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 J This application claims the benefit of priority of U.S, Provisional Application no. 63/090902, filed October 13, 2020, and incorporated herein by reference in its entirety,

REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically as a text file in ASCH format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 12, 2021, is named “20-1581 -WO_ Sequence- Listing_ST25.txt” and is 19 kb in size.

FIELD

[0003} The present disclosure provides a method for treating or preventing IgA nephropathy in mammals using thiol-containing molecules. The present disclosure also provides a method and kit for screening therapeutic agents for treating or preventing IgA nephropathy.

BACKGROUND

[0004} IgA nephropathy (IgAN), also known as Berger’s disease, is the most common form of glomerular nephritis, affecting 40-50% of all biopsy diagnoses in Asia. In most IgA nephropathy cases, the disease slowly progresses while causing gradually decreasing kidney function, with poorer outcomes among Asian patents. It is estimated that up to 30-40% patients will eventually develop end-stage renal disease (ESRD) that requires renal replacement therapy, including dialysis and/or kidney transplantation. IgA nephropathy occurs where polymeric immunoglobulin A (IgA) is deposited in the glomerular mesangium of the kidney as a manifestation of an underlying autoimmune disorder. IgA deposits cause chronic inflammation of the kidney and impairment of glomerular filtration, leading to renal insufficiency of patients. These patients experience buildup of fluid and toxic waste in the body, which may cause edema in the patient’s extremities, high blood pressure, electrolyte imbalance, and even severe life-threatening conditions such as multi-organ failure. Because the exact cause of IgA deposition in the glomerulus remains unknow n , there is no specific treatment for IgA nephropathy at the current time. Instead, following diagnosis, conventional therapy involves treatment with blood pressure-control medications. In some rapidly progressive forms of IgA nephropathy, immunosuppressant therapies such as the use of corticosteroids are prescribed. However, clinical trials of these aggressive treatments has not reached definitive conclusions about treatment benefit due to an increased risk of serious adverse events associated with corti costeroid usage. Despite the prevalence of IgA nephrophathy, the molecular mechanisms underlying IgA deposition in the glomerulus remain elusive and specific treatments of the disease are currently unavailable. Hence, there is an unmet need for new treatments that can effectively alleviate IgA nephropathy and prevent or delay onset of complications associated thereof. Further, there is a need for methods for treating or preventing IgA nephropathy as well as methods and kits to facilitate the development and the preclinieal validation of new therapeutic agents for treatments and for preventing IgA nephropathy.

SUMMARY

[0005] The present disclosure concerns methods of treating or preventing IgA nephrophathy as well as in vitro models associated with drug discovery. It has been determined that particular thiol-containing compounds can inhibit aberrant poly-IgA formation without disrupting normal protein functioning. These compounds present a promising path towards further understanding and treating IgA nephropathy.

[0006] One aspect of the present disclosure is a method of treating or preventing IgA nephropathy in mammals, the method including administering a therapeutically effect amount of a thiol-containing molecule or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

[0007] In particular embodiments, the thiol-containing molecule is cysteamine, 2-((3- aminopropyl)amino]ethanethiol, or A-acetylcysteine.

[0008] In another aspect, the present disclosure provides for a method for inhibiting poly-IgA formation, the method comprising administering to a solution of IgA monomers a thiol containing molecule.

[0009] In another aspect, the present disclosure provides for a method for screening for therapeutic agent candidates, the method comprising; providing a sample comprising poly-IgA; admixing the sample with a therapeutic agent candidate: determining the percent conversion of poly-IgA into monomeric-lgA.

[0010] In another aspect, the present disclosure provides for a kit for screening candidate therapeutic agents, the kit comprising; a container comprising a predetermined amount of poly-lgA, wherein the poly-lgA is provided as a solution, suspension, or solid; one or more optional buffers; and instructions for use of the kit.

[0011] Other aspects of the disclosure will be apparent to those skilled in the art in view of the description that follows,

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1: IgA nephropathy patients’ plasma contains poly-lgA complexes that are susceptible to reducing agent tris (2-carboxyethyI)phosphine (TCEP). A. Pooled plasma samples were collected from IgA nephrophathy patients (n=10), and total IgA l was extracted using jacalin-coBjugated column. The extraction was then resolved by SEC, which was calibrated against molecular weight standards (arrowheads: kDa vs elution volume along ,r- axis). The IgA contents formed one major peak of mono-IgA, preceded by several overlapping minor peaks of poly-lgA and dimeric slgA. B. The poly-lgA fraction was subsequently analyzed by Western blotting with ami-human IgA heavy chain antibody under either reducing (R: with TCEP) or non-reducing (NR: without TCEP) condition. With the addition of TCEP, the -600 kDa poly-lgA complexes were reduced to a -65KDa band of IgA heavy chain (IgAH).

[0013] FIG. 2 Inter molecular disulfide bond(s) involved in the self-aggregation of recombinant poiy-rlgA. A. Recombinant human and rat IgA mimetics are comprised of the CH2-CH 3-Tailpiece (TP) segment of IgA heavy chain (Left), Like native IgA hea vy chain, the mimetics form a duplex that is referred to as mono-rlgA. Transmission electron miscroscopy (TEM) images confirmed rat rlgA duplexes in donut-like appearances (right). B. Rat rlgA was resolved by size-exclusion chromatography (SEC) with a dear separation of its poly- and mono-rlgA contents, C. SDS-PAGE results confirmed the presence of disulfide connections among self-associated rlgA in poly-rlgA complexes (NR: non-reducing condition); Under reducing condition (R) both poly- and mono-rlgA reduced to single chains of 32kDa, D. TEM images of poly-rlgA SEC fraction showed rlgA aggregates (left: arrowheads): High magnification images (middle panels) show structures with multiple circular voids of monomeric rlgA units, in contrast io mono-IgA that all appeared as single donut-like structures (arrowheads). Scale bar; 10 rnn. [0014] FIG. 3: Mutagenesis analyses of cysteine~31.l and cysteine-47.l regarding formation of intermoleenlar disulfide bond. A-C. SEC analyses of rfgA in the presence of reducing agents of TCEP, dithioreitol (DTT) or glutathione, respectively. D-F. SEC tracing of C471S, C311/47 IS, and C31 1 mutations of rlgA as compared to wild-type, respectively. The bar graphs show quantitation of poly- vs. mono-rigA contents based on area under the curve (AUC). G. Comparing HEK293 cell-produced human rlgA (hu-rlgA) wild-type and C471S mutant in terms of their poly-hu-rlgA contents as revealed by SEC.

[0015] FIG. 4: Cysteamine reacts with amino acid cysteine or free cysteine in protein. A. Drug action of cysteamine in treatment of cystine crystals in cystinosis via formation of cysteine-cysteaniine (soluble) mixed disulfide. B. Both human cell-produced endogenous cysteamine and cysteamine drug interact with susceptible cysteine residues in proteins, such as Cys471 in human IgAl, as a form of post-translational modification (PTM). C. Another aminothiol drug amifostine (brand: Ethyol) is a prodrug with its active metabolite WR-1065 capable of interacting with small thiol molecules as well as unpaired cysteine residues in proteins.

[00 16] FIG. 5: Cysteamine treatment of either recombinant rlgA or native human IgA ex wro lowers poly-IgA contents. A. Cysteamine treatment of rat rlgA (produced by E. coll) showed greatly reduced poly-rlgA peak in SEC tracing. B. Human recombinant rlgA (hu-rlgA) produced by HEK293 cells has a lower level of poly-rlgA as compared to A. Treatment with cysteamine further lowered the poly-rlgA peak in SEC tracing. C. Using total IgAl extracted from IgA nephropathy patients, ex vzro treatment with cysteamine also lowered poly-1 gAl levels. D. SDS PAGE analyses showed dose-dependent response of poly- rlgA reduction by cysteamine. Still, the reactions produced duplex mono-rigA with two rlgA chains remained connected. In contrast, TCEP completely separated paired rlgA heavy chains and resulted in single chain rlgA-Fc. E, and F. In a dose-dependent manner, two additional aminothiol drugs, WR-1065 (E.) and A-acetylcysteine (NAC) (F.) also lowered the level of native human poly-IgA 1 contents ex vivo as shown by SEC.

[0017] FIG. 6. In vivo treatment of rats and mice with cysteamine lowers IgA deposition in the kidney in injection-induced IgA nephropathy models. A-C. Rat model treated with cysteamine. A. In a rat IgA deposition model, rats received a daily dose of cysteamine, or buffer control, followed by an injection of recombinant rat rlgA for 5 consecutive days. B, Representative immunoflnoresence images showed prominent rlgA deposition in glomeruli (Glom: arrowheads) in rats treated with buffer (n=3), in contrast to weaker deposits in cysteamine-treated rats (n= 3). C. Quanti tation of deposi t in glomeruli between control- and cysteamine-treatment groups was compared by t-test (MeanTSEM: I384.i-.290 vs. 523±76, n~3 in each group). D. Mouse model of IgA deposition from injection of human IgAl purified from human plasma. Each mouse was injected with a single dose of purified human IgAl two hours after pretreatment with either cysteamine or buffer control.

E. The buffer control group of mice (n=6) had prominent IgAl -deposition in glomeruli (Glom: arrowheads). In contrast, pretreatment of the mice with cysteamine (n=6) greatly reduced IgA l deposition. F. Quantification of glomerular IgA l intensity between buffer-and cysteamine-treatment groups (Mean±SEM: 2293±163 vs. 870*193, n-6 in each group). Scale bar: 50μm.

|0018j FIG. 7: Individual IgA nephropathy patients’ poly-IgA complexes disassembled by reducing agent Plasma samples were collected from eight IgA nephropathy patients. Fallowing purification of total IgAl by Jacalin beads, poly-IgA 1 complexes were extracted by SEC. Purified complexes were treated in the presence or absence of 2- mercaptoethanol and were then resolved by SDS PAGE and probed by anti -IgA Western blotting. In all samples, with the absence of 2-mercaptoethanol (NR), poly-lgAl appeared at "600 kDa.

With 2-mercaptoethanol (R), a single ~-65 kDa IgAl band corresponding to IgAl heavy chain was observed for each sample.

DETAILED DESCRIPTION

[0019] IgA nephropathy is the most common form of primary glomerulonephritis and a leading cause of end-stage kidney disease (ESKD). IgA nephropathy is thought to result from improper polymerization of IgA proteins, resulting in buildup in the kidneys through chronic deposi tion of poly-IgA complexes in the glomerular mesangium, causing inflammatory injuries to the kidney. It is generally believed that certain forms of IgA molecules in blood circulation are prone to aggregate into poly-IgA complexes, either through serf-association (see, e.g., FIG. 1- FIG. 4) or through anti-glycan antibodies against O-glycosylated epitopes on IgAl . If not cleared by the liver promptly, poly-IgA can deposit in the mesangial areas of the kidney glomerulus. In this disclosure, experiments were conducted that demonstrate that an amino acid (known as the penultimate Cys471 residue ) on the “tail piece” segment of the IgA heavy chain can form intermolecular disulfide bridges between IgA molecules (FIG. 6, FIG. 7), promoting the formation of poly-IgA.

[0020] It was noted that high molecular weight poly-IgA complexes contain aberrant 0- linked glycosylation of the hinge region of IgAl heavy chain. Also, glycoforms with reduced galactose contents are associated with higher incidence of IgA nephropathy. In-solution X- ray scattering data showed that IgAl is prone to non-specific self-association when levels of O-galactosylation is reduced, in keeping with the notion that O-glycans protect IgAl from self-aggregation, and/or adhesion to matrices. It was also suggested that these aberrant glycoforms, referred to as galactose-deficient IgAl (Gd-IgAI), are antigenic. In IgA nephropathy patients, anti-Gd-IgA I autoantibodies of IgG or IgAl can be detected, and these IgG-IgA I and IgM-IgAl antibody-antigen pairs may lead to the formation ofpoly-IgA immune complexes in circulation that are susceptible to renal deposition.

[0021] Besides glycosylation on IgAl hinge segment, another important feature of IgA molecule is that both IgA l and IgA2 isotypes have secretory and non-secretory forms. Secretory IgA (slgA) has two IgA monomers linked by two additional polypeptide subunits, namely the J-chain and the secretory component (SC). It is important to note that J-chain and SC form cysteine-to-cysteine disulfide bridges with IgA heavy chain in the slgA configuration. Specifically, J-chain’ s Cysl4 and Cys68 residues form two separate disulfide bonds with Cys47l in the so-called secretory tailpiece (tp) of IgA heavy chain to bridge two IgA molecules. SC, on the other hand, forms disulfide bond with Cys311 in CH2 domain of IgA heavy chain. In non-secretory IgAl monomer, which predominates in plasma, Cys311 and Cys471 residues are not connected to SC and J-chain, and therefore maintain their reduced and free forms. Under oxidative conditions, these free cysteines are prone to form disulfide bonds with other cysteine residues that potentially coalesce IgA into high-order molecular aggregates. Our study investigated the propensity of free Cys311 and Cys471 of human IgAl in promoting self-aggregation, as well as therapeutic means to disaggregate IgA complexes using thiol-reactive drugs.

[0022] There are a wide range of systemic diseases and localized conditions tha t can cause kidney dysfunctions and diseases. In general, antioxidants are known to be protective against oxidation and inflammation, which can attenuate nephrotoxicity. However, and without wishing to be bound by theory, this is a distinct protective mechanism from the mechanism- of-action of cysteamine, WR-1065 (also referred to as 2-[(3-aminopropyl)amino]ethanethiol), or A-acetylcysteine as disclosed herein in disassembling poly-IgA complexes and therefore reducing IgA deposition in the kidney, which is the root cause of IgA nephropathy. The experimental data disclosed herein surprisingly demonstrate that the thiol group of cysteamine does not act as a mere redox reactant, but rather specifically reacts with Cys-471 residue of the IgA heavy chain, and thereby prevents it from forming unwanted intermolecular disulfide bonds in aggregating IgA. Similarly, it is demonstrated that two other thiol-reactive compounds, W R- 1065 and N-acetyicysteine ( NAC), are also surprisingly effective in reducing pathogenic poly-IgA levels and therefore represent promising therapeutic agents in the treatment of IgA nephropathy.

[0023] As used herein, treating IgA nephropathy may refer to therapy that removes or reduces the amount of existing poly-IgA aggregation in kidney tissue. Additionally or alternatively, treating IgA nephropathy may refer to preventing additional deposits of poly- IgA from forming in kidney tissue, or preventing poly-IgA formation.

[0024] This is in contrast to reducing thiol compound glutathione that showed relatively modest effects on poly-IgA levels when used with comparable concentrations, suggesting that different thiol compounds have different potency against poly-IgA aggregates. Therefore, selecting compounds with desired reducing efficacy is critical for treatment use, as TCEP and DTT were shown below to be too potent in our ex vivo study that also inadvertently dissociated normal pairing of IgA heavy chains, while cysteamine, WR-1065 and tv- acetylcysteine did not cause unwanted disruption of natural IgA! assembly (comprised of two heavy chains and two light chains connected via disulfide bonds).

[0025] As discussed herein, several suitable tih.iol-contain.ing molecules have been identified, wherein the thiol -containing molecule is understood to be a molecule that comprises at least one thiol (i.e. -SH) moiety. Critically, the thiol-containing molecule must have sufficient activity' to inhibit poly-IgA function, but not interfere with normal IgA functionality.

Surprisingly, as disclosed herein, a particular class of compounds that meets these criteria has been identified. Accordingly, in certain embodiments as otherwise described herein, the thiol-containing molecule comprises at least one amino group. For example, in particular embodiments, the thiol-containing molecule comprises one or two amino group (e.g., wherein each amino group is either primary or secondary).

[0026] The amino groups as otherwise described herein may be neutral (e.g., -NTI; or -Nil-) or hypervalent as a suitable salt (e.g.. -W or -NHs-""). For example, the thiol-containing molecule may be present as a pharmaceutically acceptable salt thereof wherein one or more of the amino groups are optionally hypervalent. Additionally or alternatively, a carboxy group, if present, may be protonated and neutral, or deprotonated as a monoanionic moietiy. [0027] In particular embodiments, the thio I -containing molecule lias one or two thio! groups. For example, in certain embodiments, the thioi-containing molecular comprises exactly one thiol.

[0028] The proximity of the amino group and the thiol group may play an important role in drug functionality. Accordingly, in certain embodiments as otherwise described herein, a thio! of the thioi-containing molecule is bound to at least one amino group through an ethylene bridge (i.e.. a divalent C: alkyl group). In certain embodiments, the ethylene bridge is unsubstituted. In other embodiments, the ethylene bridge may be substituted by a carboxy group.

[0029] Accordingly , in certain embodiments as otherwise described herein, the thioi- containing molecule is selected from the group consisting of cysteamine, WR-1065 (i.e,, 2- [(3-aminopropyI)amino]ethanethiol), and A-acetylcysteine (i.e., A-acetyl-L-cysteine). For example, in particular embodiments, the thioi-containing molecule is cysteamine or A- acetylcysteine (NAC).

[0030] IgA may be either native IgA purified font plasma, or synthetic analogs in the form of rlgA that contains the Fc segment of IgA heavy chain. Both native and synthetic analog IgAs have a natural tendency to form poly~lg.A aggregates in solution.

[0031] To counter this aberrant polymerization, ex vivo studies discussed in the Examples below have surprisingly shown a dose dependent sensitivity of poly-IgA, either in the form of native or synthetic analogs such as recombinant IgA/rlgA, to reducing agents, such as TCEP (tris(2-carboxyethyl)phosphiiie) as well as other thioi-containing molecules, such as cysteamine. Cysteamine is of special interest as it is already an FDA approved drug to treat cystinosis by dissolving cystine crystals. Accordingly, in certain embodiments, the method of treating or preventing IgA nephropathy comprises the thioi-containing molecule binding with an IgA monomer, while having desired potency that does not disrupt normal assemblies of IgA and other nature protein complexes to cause adverse drug effects.

[0032] As disclosed herein, it has been found that certain thioi-containing molecules, such as cysteamine, are able to convert aberrantly formed poly-IgA to monomeric IgA (comprised of two heavy chains and two light chains) in a dose-dependent manner, and can also effectively reduce, and/or prevent, IgA aggregation and thus is useful for treating IgA nephropathy. Additionally, it has been determined that intravenous injection of poly-rlgA, purified through size-exclusion chromatography, in rats causes glomerulus deposition of IgA. In contrast, administration of cysteamine prior to intravenous poly- IgA injec tion prevented IgA deposition in kidney glomeruli. The resulting poly- IgA treatment produced an animal model useful for evaluating IgA nephropathy treatments. Furthermore, the animal model established that certain tluol-coiitaining molecules can be useful for treating or preventing IgA nephropathy.

[0033] The thiol-containing molecule as otherwise described herein may be administered to a wide variety of mammalian subjects. For example, in certain embodiments, the mammal is selected from the group consisting of human, rat, mouse, dog, monkey, chimpanzee, and rabbit. [0034] In another aspect, the present disclosure provides for a method for inhibiting polylgA formation, the method comprising administering to a solution comprising IgA monomers a thiol-containing molecule. For example, the thiol-containing molecule as otherwise described herein.

[0035] In another aspect, the present disclosure provides for a method for screening for therapeutically effective agents, the method comprising: providing a sample comprising poly-IgA; admixing the sample with an agent of interest; determining the percent conversion of poly-IgA into monomeric -IgA.

[0036] For example, the agent of interest may be a thiol-containing molecule as otherwise described herein. Alternatively, the method for screening as otherwise described herein may, in certain embodiments, be utilized to identify novel classes of molecules that are therapeutically effective. The sample can include recombinant poly-IgA, poly-IgA obtained from the bodily fluid, e.g., blood or blood component (e.g., plasma) of a patient or generated from IgA by subjecting IgA to a procedure, e.g., chemical oxidative procedure. The sample can include any suitable aqueous liquids such as one or more buffers or saline to suspend, admix or dissolve the poly-IgA.

[0037] In another aspect, the present disclosure provides for a kit for screening candidate therapeutic agents, the kit comprising: a container comprising a predetermined amount of poly-IgA, wherein the poly-IgA is provided as a solution, suspension, or solid; one or more optional buffers; and instructions for use of the kit.

[0038] Suitable examples of buffers include saline, citrate-based buffer, and phosphate-based buffers, such as PBS. Suitable examples of containers include vials or bottles. The poly- IgA may be prepared from native IgA purified from plasma,, or synthetic analogs in the form of rlgA that contains the Fc segment of IgA heavy chain. Both native and synthetic analog IgAs have a natural tendency to form poiy-lgA in solution. Purified poly-IgA may be purified by any method as known in the art, such as through size exclusion chromatography. The poly-IgA included in the kit as otherwise described herein may be provided in any suitable form. For example, in certain embodiments as otherwise described herein, the poly-IgA is dissolved in solution, for example, a buffer solution. In other embodiments, the poly-IgA is at least partially precipitated out of solution, and so is provided as a suspension. In further embodiments, the poly-IgA may be isolated as a solid powder. In such embodiments, the kit may further comprise a buffer solution that may be used to reconstitute the poly-IgA for use. Alternatively, the kit may comprise instructions for preparing a suitable buffer solution for use of the poly-IgA.

[0039] As discussed further in the Examples, it has been determined that the effectiveness of certain agents for preventing or treating poly-IgA deposition may be observed in mammals. For example, in certain embodiments as otherwise described herein, the method further comprises determining the amount of poly-IgA deposition. This may be conducted through techniques known in the art. For example, in particular embodiments, the determining the amount of poly-IgA deposition is performed through kidney imaging, optionally through immunofluorescence imaging.

[0040] The compounds as otherwise described herein may not only be useful in the treatment of poly-IgA deposits, but may also function to prevent such deposits as well. Accordingly, in another aspect, the present discl osure provides for a method of preventing IgA nephropathy in a mammal, the method comprising administering a therapeutically effect amount of a thiol- containing molecule, or a pharmaceutically acceptable salt thereof For example, the thiol- containing molecule may be that as otherwise described herein.

[0041| Advantageously, cysteamine is readily available in the forms of oral medication (in capsule and extended release formulations), and also in eye drops. In certain embodiments as otherwise described herein, cysteamine is provided in a commercially available form, for example, those sold under the trade names Cystagon^® (Mylan), or Procysbi^® (Horizon Therapeutics), or Cystaran^® (Leadiant Biosciences).

[0042] WR-1065 (2-[(3-aminopropyl)amino]ethanethiol) is a metabolite and active form of Amifostine (cthiofos: brandname: Ethyofo), an approved drug for reducing renal toxicity with chemotherapy and for moderate to severe xerostomia from radiation therapy.

[0043] A-acetylcysteine is available as a common oral supplement and used to treat acetaminophen overdose, and may be obtained from Sigma-Aldrich.

[0044] The thiol-containing molecule according to the present disclosure may be neutral form, or may be a pharmaceutically acceptable salt thereof. For example, in certain embodiments as otherwise described herein, the thiol-containing molecule may be a salt or ester or derivative. Examples of suitable salts include those formed with organic or inorganic acid, such as salts of acetate, tartrate, bitartrate, trifluoroacetate, lactate, maleate, fumarate, citrate, methanesulfonate, sulfate, phosphate, nitrate, or chloride. In particular embodiments, the thiol- containing molecule is a bitartrate salt. For example, in particular embodiments, the cysteamine is cysteamine bitartrate.

[0045] The thiol-containing molecule described herein may be administered orally or intraveneously in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The pharmaceutical compositions described herein may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft: capsules, or syrups or elixirs.

[0046] Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be ancoated or they may be coated by known techniques, for example with an enteric coating. In some cases such coatings may be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0047] Formulations for oral use may also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Formulations for ora! use may also be presented as lozenges.

[0048] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from faty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from faty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0049] The methods of the present disclosure involve the administration of an effecti ve dose of a thiol-containing molecule to treat IgA nephropathy in mammals such as humans. In certain embodiments as otherwise described herein, the thiol-containing: molecule can be administered in a daily amount ranging 0.1 mg/kg to 400 mg/kg, or 1 mg/kg to 400 mg/kg. For example, in certain embodiments, the thiol-containing molecule can be administered in daily amount ranging from 5 mg/kg to 80 mg/kg. In other embodiments, the thiol-containing molecule can be administered In a daily amount ranging from 10 mg/kg to 250 mg/kg, or 20 mg/kg to 250 mg/kg, or 40 mg/kg to 250 mg/kg. For example, in particular embodiments, the thiol-containing molecule is cysteamine or xV-acetylcysteme, and is administered in a daily amount in the range of 1 mg/kg io 150 mg/kg, for example, in the range of 2 mg/kg to 100 mg/kg, or 5 mg/kg to 80 mg/kg, or 5 mg/kg to 60 mg/kg, or 10 mg/kg to 50 mg/kg. It is to be understood that the milligram dosage quoted reflects the equivalent milligrams of pure thiol- containing compound (i.e. , without inclusion of any anions or salts in the molecular weight). |0050| In certain embodiments as otherwise described herein, the dose of the thiol-containing molecule can be administered one or more times per day, such as one time per day, two times per day, three, four, or six times per day. In certain embodiments as otherwise described herein, the thiol-con taining molecule or the composition comprising the thiol-containing molecule is administered for any s uitable period of time. For example, the thiol-containing molecule or the composition comprising the thiol-containing molecule may be administered for a period of at least three weeks, or a period of 4-6 weeks, or for a period of at least 4 weeks, 6 weeks, 8 weeks, 12 weeks, or at least 24 weeks.

[0051} Examples of methods and systems are described herein. It should be understood that the words “exemplary,” “example,” and “illustrative,” are used herein to mean “serving as an example, instance, or illustration.” Any embodiment or feature described herein as “exemplary,” “example ” or “illustrative,” is not necessarily to be construed as preferred or advantageous over other embodiments or features. Further, the exemplary embodiments described herein are not meant to be limiting. It will be readily understood that certain aspects of the disclosed systems and methods can be arranged and combined in a wide variety of different configurations.

EXAMPLES

Methods

Recombinant construction of rat and human IgA mimetics

(0052} DNA sequence (SEQ ID NO.: I ) that encodes wild-type rat IgA-Fc segment of CH2- CH3-TP (SEQ ID NO.: 2) was cloned into PET30a vector (Invitrogen, Carlsbad, CA) with an N-temiinus 6xHis-tag using standard procedures. Corresponding point mutations of C471 S (SEQ ID NOs.:3 and 1 l((rat and human, respectively), C31 IS (SEQ ID NO.:5)(rat) and C311/47 IS (SEQ ID NO.:7)(rat) were generated by site- directed mutagenesis using standard procedures. Human IgAl-Fc CH2-CH3-TP cDNA (SEQ ID NO.:9) and its mutant for C471S (SEQ ID NO.: 11) were fused to sequences encoding IL-2 signal peptide and 6xHis-tag at the 5 ’-end in pcDNA3 vector (Invitrogen, Carlsbad, CA) using standard procedures. Rat IgA analogs were expressed in BL21 DE3 strain of £. co/i and recombinant protein were purified from the bacterial lysate using Histrap HP columns (GE Healthcare) by running fast protein liquid chromatography (FPLC). Human recombinant IgA mimetics were produced from human embryo kidney (HEK293) cel ls by transfection of the pcDNA3 plasmids. Recombinant IgA proteins expressed by the cells were secreted into the culture medium, and they were subsequently purified by Histrap HP columns via running FPLC following standard procedures, such as those discussed in Liu. “Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation.” Kidney Int. 94(1 ).T 14-125 (2018).

Native IgA.purificatfon.ftom hunian, serum

[0053] IgAl from IgA nephropathy patient sera was purified by Jacalin (T hermo Scientific, USA)-directed affinity chromatography. Poly-IgAl contents were further enriched via running Superdex 200 Increase gel filtration molecular sieve (GE Biosciences) and collecting high-molecular weight fractions from an AKTA protein purification system (GE Biosciences). Human serum from healthy donors was purchased from Sigma- Aldrich and total IgAl was also purified by running Jacalin-directed chromatography. Purified total IgAl was used for injecting mice.

Analysis of intennolecular disulfide bond formation in IgA self-aggregates: determination of poly- and monomeric IgA content

[0054] The overall complex size of IgA aggregates was determined either by size-exclusion chromatography (SEC: Superdex 200 Increase 10/300 by GL Cytiva), or by SDS-PAGE. For evaluating the involvement of intennolecular disulfide connectivity in IgA complexes, IgA samples were treated with either reducing agents, such as DTT, TCEP or reduced glutathione (Sigma), or interventional drugs, such as cysteamine (Sigma), WR-1065 (Sigma) and N- Acetyl-L-cysteine (VWR Chemicals) in phosphate-bufiered saline (PBS ) pH7.4. Before running on SEC, samples were treated with the drugs at indicated concentrations at 37ºC for one hour.

[0055] In the meantime, the SEC column was also equilibrated with PBS supplemented with the reducing agent at the same concentration. For running SEC, 100 pL of the recombinant IgA (concentration varies between I mg/mL and 8 mg/mL) or purified native human IgAl (-1 mg/mL) was loaded to the column and UV absorption was recorded following elution. Recombinant IgA typically formed two elution peaks: a minor peak of -800 kDa poly-rlgA followed by a major peak of -170 kDa of monomeric rlgA. The ratios between poly-rlgA and mono-rlgA were calculated as between the corresponding areas under the curve ( AUC), and the treatment effect of the thiol agents was judged by the reduction of the poly-rigA level with a compensatory increase of the mono-rlgA content. With regard to native human IgA 1, SEC showed three partially overlapping peaks of poly-, di- and mono-IgAl, and treatment effect was judged by the reduction of the poly-Ig.Al level with a compensatory increase of the mono-IgA 1 content.

SPS-PAGE and. Western, Blotting

[0056] Proteins in sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) with or without TCEP (for reducing or nonreducing condition, respectively) were resolved by 4~ 12% SDS- PAGE (Bio-Rad Laboratories), IgA bands were subsequently visualized either by staining with GelCode Blue (Thermo Fisher Scientific), or by Western blotting on PVDF membrane. For Western blotting, 5% non-fat milk was used in blocking for one hour at room temperature. The membrane was then incubated with mouse anti-Histag antibody (Thermo Scientific), or HRP-conjugated goat anti-rat IgA a-chain antibody (Cat. ab97185. Abeam, UK), or goat anti-human IgA HRP antibody (Cat:2050-05, Southern B iotech) for detecting rat or human IgA (Fc), respectively. The membrane was developed using the Clarity™ ECL substrate (Bio-Rad Laboratories, CA, USA).

Transmission Electron Microscopy (TEM) Analyses

[0057] TEM analyses of the structures of poly-rigA and mono-rlgA were conducted following a. standard negative staining protocol. In brief, purified poly-rigA or mono-rlgA was diluted in PBS to a concentration of 100 pg/ml. A 10 pl droplet was applied to a glow - discharged carbon-coated copper grid and allowed to sit for 1 min. The grid was washed by dipping in two separate drops in water followed by two drops in 2% uranyl acetate (Electron Microscopy Sciences). Grids were examined at the Northwestern Electron Probe Instrumentation Center (EPIC) using Hitachi HT - 7700 Biological S/TEM Microscope.

Cysteamine treatment of IgA deposition in rat and mouse model

[0058] T o establish the passive rat model, 5mg/kg.BW recombinant rat rlgA, which contained a traction of poly-rigA, were z.v. injected daily to six 14-weeks old male Wistar rats (Charles River Labs, USA) for 5 consecutive days. Every day, three rats in each group had recei ved a subcutaneous dose of either 250mg/kg cysteamine, or buffer control, two hours before the rlgA injection. Twenty-four hours after the last injection of rlgA, kidneys were collected for immunofluorescence detection of deposits with goat-anti-fat IgA antibody (CatSTAR11 1, Biorad laboratory). Similarly, a passive IgA nephrophathy mouse model was established by injecting 35mg/kg purified human IgAl in BALB/c mice (Charles River Labs). Two hours before IgAl injection, six mice in each experimental group had each recei ved a pretreatment dose of either 200mg/kg cysteamine, or PBS, via subcutaneous injection. Two and a half hours after human IgA 1 injection, kidneys were harvested, and specimens were stained with FTTC-conjugated anti-human IgA antibody (Cat:2050~02, SouthemBiotech). Plasma samples were collected at 0.5h. Ih and 2h after IgAl injection of all mice.

Immunofluorescence staining

[0059] Frozen tissues were sectioned at 4 pm for IgA detection using goat anti-rat IgA (Cat.STARl 1 1 , Bio-Rad Laboratories) at 1:100, or anti-human IgA antibody (Cat:2050-02, Bio-Rad Laboratories) at 1 :80 dilution. Anti-collage IV a I (Cat:NB 120-6586, Novus) at 1 :500 dilution, rat anti-mouse CD31 (Cat:553370, BD Biosciences) at 1:100 dilution and DAP1 were used as counterstaining. Immunofluorescence images were captured by Nikon Ti2 Widefield microscope. The mean immunofluorescence intensity per glomerulus area was derived from 15 glomeruli per kidney section assisted by Image J software.

Statistical analyses

[0060] Data are displayed graphically, and statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software). Group data are reported as mean± SEM. Significance between two groups was determined by t-test. Significance was accepted when p -values were

0.05.

Example 1 : High motecular weight I gA co m plexus extracted from I g A nephropathy patients- plasma contain intermolecnlar disulfide connections

[0061] In this Example, it was sought to isolate high molecular weight IgA complexes, such as poly-IgA, from plasma of IgA nephropathy patients. A standard workflow was followed to purify total IgAl from pooled plasma using a jacalin-conjugated affinity column, and then subjected the extraction to size-exclusion chromatography (SEC). IgA monomers formed the dominant peak of - 160 kDa, preceded by dimeric slgA of -350 kDa (Fig. 1, part A). Further ahead, poly-IgA formed additional overlapping minor peaks at. >670 kDa ( Fig. 1, part A). By running SDS-PAGE of the poly-IgA fraction under both reducing and nonreducing conditions, it was determined that the molecular complexes of IgA were connected through disulfide bridges (Fig. 1, part B). To be certain of broad presence reduction-sensitive IgA complexes, another cohort of patients was recruited. IgA 1 complexes were extracted from individual patients by SEC and analyzed the contents by SDS-PAGE. These samples showed varying levels of high molecular weight IgA 1 contents that could be dissociated by reducing agents (Fig. 7).

Example 2; Mutagenesis of recombinant IgA mimetics identified penultimate residue cvs teine-471 in promoting IgA aggrega tion

[0062 J To further investigate specific cysteine residue(s) involved in IgA complexes, expression vectors were constructed to produce recombinant IgA (rig A) Fc segments of rat and human sequences (Fig. 2, part A) as discussed above. Rat IgA analogs were expressed in BL.21 DE3 strain of A. co/z and recombinant protein were purified from the bacterial lysate using Histrap BP columns (GE Healthcare) by running fast protein liquid chromatography (FPLC). Human recombinant IgA mimetics were produced from human embryo kidney (HEK293) ceils by transfection of the expressing plasmids. Recombinant IgA proteins expressed by the cells were secreted into the culture medium, and they were subsequently purified by Histrap HP columns by FPLC using standard procedures. Like native IgA heavy chain, these single-chain rlgAs also folded into duplex, referred to as mono-rlgA in keeping with tradition. Rat rlgA was produced from bacterial expression as discussed above. Rat rlgA was resolved by SEC (Fig. 2, part B), showing a major peak of mono-rlgA duplex at -64 kDa, preceded by a minor peak of poly-rlgA at >500 kDa. SDS-PAGE results further confirmed intermolecular disulfides in connecting rlgA units (Fig. 2, part C). Transmission electron microscopy (TEM) showed interconnected rlgA structures in high-order complexes (Fig. 2, part D). To further demonstrate that poly-rlgA was also linked by disulfide bridges, reducing agents were added such as d’ris(2-carboxyethyl)phosphine hydrochloride (TCEP), 1,4-Dithiothreitol (DTT) or glutathione to rlgA. Analyses on SEC showed poly-rlgA disassembled into monomers by TCEP and DDT, and to a lesser degree, by glutathione (Figs.

3, part A-3, part C). Specifically, SEC analyses showed a concentration-dependent reduction of the high molecular weight poly-rlgA peak by TCEP. Meanwhile, there was a slight compensators' increase of the mono-rlgA content (Fig. 3, part A), as expected. [0063] Considering that the fragment contained Cys3 I 1 and Cys471 that, in the absence of J- chain and SC, were in their free forms and available tor connecting other rigA units, mutagenesis studies were conducted of these two cysteines. Cys311 and Cys471 were either individually, or together, mutated to serine (S) as discussed above. These C31 1 S (SEQ ID NO.. '6) (rat only) and C471S single mutants (SEQ ID NOS.: 4 AND 12) (mt and human, respectively), and C311/471 S double mutant (SEQ ID NO.:8) (rat only) were produced as rat rigA proteins. By examining the SEC traces of these mutants, it was noticed that, while the monomers of all variants appeared the same, there were dramatic differences in the poly-rlgA contents. Notably, the prominent high molecular weight peak for poly-rlgA completely disappeared in C471S single mutant and in C331/471S double mutant (Figs. 3, part D-3, part E). Meanwhile, C311 S-alone still had poly-rlgA contents, albeit eluted at a different time in SEC than the wild-type protein (Fig. 3, part F). In addition, SDS-PAGE results further confirmed Cys471 ’s involvement in poly-rlgA formation.

[0064] In parallel, the experiments were repeated using human rlgAl-Fc (SEQ ID NO.: 10) and its C471 S mutant ( SEQ ID NO.:12), which were produced from mammalian cell expression. Wild-type rlgAl also had both monomer and polymer contents, in contrast to C471 S showing greatly reduced level of poiy-rlgAl (Fig. 3, part G), suggesting that Cys471 promotes rigA self-association.

Example 3: Cysteamine reduces poIv-IgA levels in vit

ro

[0065] After identifying the tailpiece cysteine in connecting IgA molecules to form aggregates due to spontaneous self-association, it was sought to test interventional drugs to disassemble IgA complexes by reducing the disulfide bond on Cys471. Cysteamine (Cy), an aminothiol that can react with cysteine, is both a natural metabolite produced in mammalian cells and a clinical drug used for treatment of cystinosis. In cystinosis, cysteamine reduces the disulfide bond in cystine, which is the oxidated dimer of amino acid cysteine (Fig. 4, part A), hi the context of cysteine residues in proteins, endogenous cysteamine as well as the therapeutic drug forms mixed disulfides with susceptible cysteine sulfhydryl groups in a process called cysteamination (Fig. 4, part B). It was sought to determine whether cysteamine could reduce Cys471 -disulfides to disaggregate poly-IgA. Rat rigA (SEQ ID NO.4) was treated with various concentrations of cysteamine for one hour at 37°C in phosphate buffered saline at neutral pH and the protein complexes were analyzed by SEC. The SEC column (model: Superdex. 200 Increase 10/300 GL by Cytiva) was preequilibrated with the same buffer supplemented with the same concentration of cysteamine. 100 μL of treated rlgA was loaded to the SEC column and the run used the same phosphate buffered saline with corresponding concentration of cysteamine. As expected, the drug effectively lowered poly-rlgA levels in a dose-dependent manner, whereas the relative amounts of mono-rlgA slightly increased ( Fig. 5, part A). The result suggested that cysteamine was able to disassemble high molecular weight IgA complexes by disrupting intermolecular disulfide bond. The results are summarized in Table 1 :

Table 1

10066} Human rfgAl (SEQ ID NO.: 10) produced from mammalian expression as discussed above had a smaller fraction of poly-rlgA contents as compared to E coli produced rat rigA (compare Figs. 3, part G and 2, part B). Nevertheless, treatment of h uman rlgA with cysteamine also showed reduction of poly-rlgA levels (Fig. 5, part B). In addition, human IgA I whole molecule was purified from pooled plasma of IgA nephropathy patients and subjected the sample to different concentrations of cysteamine (Fig. 5, part C). In a dose- dependent manner, cysteamine reduced the amounts of poly-lgAI , whereas the mono- IgA I levels slightly increased in response to cysteamine, as expected. Because the dimeric slgAl peak partially overlapped with that of poly-IgAI on SEC, it is difficult to accurately assess the impact of treatment to slgA I dimers. However, as the overall shape of slgA peak remained largely unchanged, it was expected that the contents of slgA 1 remained stable. Collectively, these results indicated the structural susceptibility of Cys471 to cysteamine in poly-IgAI, in contrast to the stability of monomers. [0067] To further ascertain the tnechanism-of-action of cysteamine in targeting disulfides, nonreducing SDS-PAGE analysis was performed of rat poly-rlgA (Fig. 5, part D). The samples were pretreated with 0 to 10 mM cysteamine, or 10 mM TCEP as positive control.

As expected. poly-rlgA appeared as >250 kDa bands on SDS-PAGE. Dose-dependent response of poly-rlgA to cysteamine was evident, with majority of the protein running at ~64 kDa of disaggregated monomers. Meanwhile, lOmM TCEP treatment resulted in further reduction of the molecular weight to -32 kDa as a single-chain protein, indicating normal disulfide bridges between IgA heavy chains being disrupted. These results suggested that modest reduction activity of cysteamine, as compared to TCEP, was effective in reducing vulnerable imerrnolecular disulfides between poly-lgA units, while leaving normal disulfides between paired IgA heavy chains intact.

[0068]

Example 4; WR-1065, the active metabolite of radioprotective drug amifostine/ethiofos ( Ethy ol), reduces poly-lgA lev el s etc w ro ,

[0069] Another thiol-based drug, amifostine (Brand: Ethyol) ( Fig. 4, part C), is a clinical radioprotector and cytotoxic chemo-protector. Its active metabolite WR- 1065 is an aminothiol that, like cysteamine, can react with cysteine, as well as detoxify non-protein metabolites. Similarly, WR-1065 was added to reactions with total IgA 1 extracted from human sera. As expected, SEC analyses showed reduction of poly-lgA levels following treatment (Fig. 5, part E).

Example 5: N-Acetyl-L-cysteine also reduces poly-lgA levels ex vivo.

[0070] A-Acetyl-L-cysteine, also known as A- Acetylcysteine (NAC), is a dietary supplement. It is also a medication that is used to treat paracetamol (acetaminophen) overdose, and other diseases. For instance, inhaled A- Acetylcysteine is used for mucolytic therapy by breaking down protein disulfide bonds, and therefore reducing the viscosity of mucus. Similar io amifostine in our ex vivo SEC analyses (Fig, 5, part E), when A-Acetylcysteine was used to treat total IgAl purified from human sera, it lowered poly-lgA levels in a dose-dependent manner (Fig. 5. part F).

Example 6: In vivo treatment with cysteamine reduces glomerular mesangial IgA deposition in murine models [0071] It was sought to test the In ww efficacy of cysteamine in a rat model of IgA deposition. A passive induction model was used with 5 daily z. v. injections of purified recombinant rat rlgA (SEQ ID NO/.2) in rats (Fig. 6, part A, left panel ). Rats developed prominent IgA deposition in the glomerulus (Fig. 6, part B, top panel). Meanwhile, rats that also received subcutaneous doses of cysteamine two hours pr ior to each rlgA injection (Fig, 6, part A, right panel) showed less glomerular deposition (Fig. 6, part B, bottom panel and Fig. 6, part C). In order to confirm that rlgA deposition was attributable to disulfide- connected poly- IgA. and reduction of its signals in the glomerulus by cysteamine treatment was due to disassociation of the complexes, mono-rlgA and poly-rlgA were purified separately by SEC as discussed above. As expected, injection of poly-rlgA fraction resulted in mesangial deposition signals, whereas injection of mono-rlgA fraction did not cause glomerular deposition. In establishing the passive rat model for IgA deposition, 5 mg/kg recombinant rat rlgA, which contained a fraction of poly-rlgA, was i.v. injected daily to 14- week-old male Wistar rats (Charles River Labs) for 5 consecutive days. Every day, 3 rats in each group received a subcutaneous dose of either 250 mg/kg cysteamine or buffer control 2 hours before the rlgA injection. Twenty-four hours after the last injection of rlgA, kidneys were collected for immunofluorescence detection of deposits with goat anti-rat IgA antibody (catalog STAR111, Bio-Rad). Similarly, a passive IgA deposition mouse model was established by injecting 35 mg/kg purified human IgAl in BALBZc mice (Charles River Labs). Two hours before IgAl injection, mice in each experimental group each received a pretreatment dose of either 200 mg/kg cysteamine or PBS via subcutaneous injection. Two and a half hours after human IgAl injection, kidneys were harvested and specimens were stained with FITC-conjugated anti-human IgA antibody (catalog 2050-02, SoutheniBiotech).

[0072] Next, it was sought to examine treatment response of human IgA to cysteamine. Another passive induction model for IgA deposition was used with injections of human IgA 1 in mice. Mouse, unlike human and rat, does not have IgA Fc receptor CD89/FcaRI that can eliminate poly-IgA complexes by phagocytic cells including the macrophage, neutrophil and Kupffer cells. One-time z.v. injection in mice was performed with total IgAl purified from human plasma (Fig. 6, part D, left panel). As expected, immunofluorescence staining of kidney specimens showed glomerular deposition of human IgAl, prominently located along capillary loops and in mesangial areas ( Fig. 6, part E, left panel). In contrast, pretreatment of mice with a subcutaneous dose of cysteamine at 200mg/kg,BW two hours prior to IgA injection ( Fig. 6, part D, right panel) showed markedly lower glomerular IgA signals (Fig. 6, part E, right panel). Particularly, the broad presence of high intensity IgAl puncta mostly disappeared in the animals that received this prophylactic dose of cysteamine (Fig. 6, part Fl. Meanwhile, blood IgAl levels showed no significant difference between the two groups of mice, demonstrating specific reduction of IgA deposition in the kidney by cysteamine.

[0073] The foregoing study investigated structural features of IgA that could potential ly make it susceptible to forming high-order complexes and causing IgA nephropathy. Unexpectedly, it was discovered poly- IgA complexes isolated from patients’ plasma disassembled by reducing agents, suggesting IgA, and possibly also their non-IgA constituents, interconnected via disulfide bridges. In addition to the cysteine residues that participate disulfides between the pair of IgA heavy chains and between heavy and light chains, there are two additional cysteines, namely cysteine-311 and cysteine-471. involved in linking Secretory Component and J~chain subunits of dimeric IgA, respectively. However, because mono-IgA 1 is the main circulatory form in blood, these two cysteine residues are expected to be in their free and reduced forms, and therefore susceptible to oxidation. It was further discovered that cysteine-471 in particular can mediate aberrant self-association of IgA and promote glomerular deposition of the resulting poly-IgA complexes. It was then sought the use of aminothiol drugs, such as cysteamine and WR-1065, to stabilize mono-IgA in preventing poly-IgA-directed kidney deposition. Ex vivo and in vivo results showed that these interventional drugs were efficacious in preventing IgA seif-association and its glomerular deposition in rodent models.

[0074] Admittedly, it remains unclear as to whether the mechanism-of-action with cysteamine was through disassembling of poly-IgA l in circulation, or in renal deposits followed by accelerated clearance, or both. Critically and without being bound by any theory of any mechanisms of action, it should also be noted that cysteamine is not a sufficiently potent reducing agent to effectively break existing disulfide bonds (Fig, 5, part D). Instead, therapeutic cysteamine possibly protects free Cys471 of IgA 1 via cysteamination io lower its reactivity towards IgAl . or other plasma and matrix proteins. Generally, intermolecular disulfide reactions are stochastic, but that is facilitated by noncovalent interactions between two proteins to position a pair of cysteine residues in proximity, often at interaction interfaces, Therefore, it is conceivable that any normal disulfides that are integral parts of immunoglobulin folds, such as those between IgA heavy chains (as in Fig. 5, part D), or between the heavy chain and the light chain (not tested), could withstand cysteamine treatment In other words, aberrantly connected disulfide interactions are expected to be more susceptible to the drugs. Nevertheless, alkylating agents that form irreversible bonds with cysteine residues, such as N-ethylmaleimide or iodoacetamide, risk destabilizing the entire IgA molecule, and may not be desirable for treatment as compared to milder aminothiols such as cysteamine. With regard to the in vivo models in our study, it should be cautioned that they do not fully resemble the clinical development of IgA nephropathy, which typically follows a slow course of progression over a long period of time. This is likely due to patients’ chronic exposure to low levels of injurious poly-lgA complexes. In contrast, the passive induction models with bolus doses of exogenous IgA presented spikes of poly-lgA levels in blood, causing acute deposition in the kidney. Because mild aminothiols function through shifting the dynamic equilibrium between free and disulfide cysteines, chronic models that fully phenocopy clinical IgA nephropathy are better choices for evaluating treatment effects.

[0075] Although it is discussed that the intrinsic propensity of IgA 1 to self-aggregate via intermolecular disulfkies, the present findings do not contradict with the established ‘galactosylation-centric’ theory of IgA nephropathy. Structural evidence suggests that hypo- galactosylated IgA may be prone to self-aggregation. The study suggests that noncovalent coalescence of hypo-galactosylated IgA units in the first place could facilitate disulfide connectivity of IgA tailpiece, which in turn, enhances self-association. Similarly, the well- accepted multi-hit model for the pathogenesis of IgA nephropathy includes antigenicity of Gd-IgA in inciting auti-glycan autoantibodies in promoting IgA immune complexes. It is still plausible that these antibody-antigen interactions bring IgA together for their reactive cysteine-471 to form disulfide bridges with additional serum and/or matrix proteins.

[0076] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope being indicated by the following claims.

Claims

We claim:

1. A method of treating or preventing IgA nephropathy in mammals, the method comprising administering a therapeutically effective amount of a thiol -containing molecule, or a pharmaceutically acceptable salt thereof to a mammal in need of treatment thereof wherein the thiol -containing molecule is selected from the group consisting of cysteamine, 2-[(3-aminopropyl)amino]ethanethioI (WR- 1065), and A -acetylcysteine.

2. The method of claims claim 1 , wherein the thiol-containing molecule is cysteamine or A-acetylcy steine .

3. The method any of claim 1 or claim 2, wherein the thiol-containing molecule is cysteamine.

4. The method of any of claims 1 -3, wherein the thiol-contaimng molecule is provided as a pharmaceutically-acceptable salt.

5. The method of any of claims 1 -4, wherein the thiol-containing molecule is provided as a hydrochloride salt or a bitartrate salt.

6. The method of any of claims 1 -5, wherein the treating comprises the thiol-containing molecule binding with an IgA residue.

7. The method of claim 6, wherein the IgA monomer is human IgA 1, and binding occurs at Cys471.

8. The method of any of claims 1 -7, wherein the administering is performed one or more times during the day.

9. The method of any of claims 1 -8, wherein the thiol-containing molecule is administered in a daily amount in the range of 0.1 mg/kg to 400 mg/kg.

10, The method of any of the claims 1-9, wherein the thiol-containing molecule is administered in a daily amount in the range of I mg/kg to 200 mg/kg

1 1. A method for screening for therapeutic agent candidates, the method comprising: providing a sample comprising poly-IgA; admixing the sample with a therapeutic agent candidate; determining the percent conversion of poly-IgA into monomeric-IgA.

12. The method of claim I 1, wherein the agent is a thiol -containing molecule.

13. The method of claim 1 i or claim 12, wherein the determining is performed through size exclusion chromatography.

14. A kit for screening candidate therapeutic agents, the kit comprising: a container comprising a predetermined amount of poly-IgA, wherein the poly-IgA is provided as a solution, suspension, or solid; one or more optional buffers; and instructions for use of the kit.

15. The kit of claim 14, wherein the poly-IgA is provided as a solid, wherein the kit further comprises a buffer solution.